Laser Raman investigation of intact single muscle fibers on the state of water in muscle tissue

381

Biochimica et Biophysica Acta, 544 (1978) 381--393

© Elsevier/North-Holland Biomedical Press

BBA 28727 BOVINE A D R E N O C O R T I C A L MICROSOMAL H Y D R O X Y L A S E AND T H E R M O T R O P I C TRANSITIONS SUBSTRATE-CYTOCHROME P-450 BINDING REACTION VERSUS SUBSTRATE H Y D R O X Y L A T I O N

SHAKUNTHALA NARASIMHULU * Harrison Department for Surgical Research, University of Pennsylvania, Philadelphia, Pa. 19174 (U.S.A.)

(Received April 7th, 1978}

Summary The effect of temperature on steroid C-21 hydroxylation and substratec y t o c h r o m e P-450 binding reaction under turnover conditions (NADPH + 02 are investigated. The Arrhenius activity plot exhibited a single break, while the van 't H o f f plot of the substrate dissociation constant (Ks) exhibited four breaks between 10 and 40°C which corresponded to the characteristic temperatures of the lipids' phase transitions. Unlike the case of the K s value, the detergent Triton X-114 was without effect on the Arrhenius activity plot. This indicates that the single break in the case of the enzyme activity is distinct from b u t n o t necessarily independent of the multiple breaks in the case of the K s. At physiologic temperature and concentration of the substrate, the free energy (--9.5 kcal/mol) of the substrate-cytochrome binding reaction is more than sufficient to account for the apparent activation energy (6.6 kcal/mol) of the overall hydroxylation. This suggests that the substrate-cytochrome P-450 binding reaction has the potential of being a source of energy for the overall reaction.

Introduction The Arrhenius plots of membrane-bound enzyme activities often exhibit discontinuities at temperatures at which the lipids undergo a phase change [1--4]. It is presumed that thermally activated conformation transitions required for the functioning of membrane enzymes are mediated through a phase change in the lipids [5]. Parts o f this w o r k w e r e p r e s e n t e d at the F A S E B M e e t i n g s [ 3 3 ] . * Mailing address: P.O. B o x No. 9 7 , Bala C y n w y d , Pa. 1 9 0 0 4 , U.S.A.

382 The adrenal microsomal steroid hydroxylase, like other mammalian hydroxylases, is membrane-bound. The reaction catalyzed by the hydroxylase is a multistep process and can be viewed as consisting of three major steps: (1) the binding of the substrate to cytochrome P-450; (2) electron transport to the cytochrome, the known mediator of which is NADPH-specific flavoprotein reductase; (3) oxygen activation and substrate hydroxylation. Functional relationships between hydroxylases and phospholipids have been investigated in hepatic as well as in adrenal microsomal systems using different approaches. Reconstitution studies [6] using detergent solubilized, partially purified and homogeneous cytochrome P-450 from hepatic microsomes have demonstrated an apparent role for phospholipids in the reduction of the cytochrome. Additional evidence suggesting involvement of phospholipids in cytochrome P-450-catalyzed reactions comes from studies with partially resolved hydroxylases from bovine adrenocortical microsomes depleted of their lipids in the absence of detergents, by organic solvent extraction [7]. A second line of approach involves the study of the effect of temperature on hydroxylases in microsomal membranes. In hepatic microsomes, Arrhenius plots with a single discontinuity have been reported for cytochrome P-450 reduction [8] as well as for overall hydroxylation reactions [9,10]. In all of these instances, the discontinuities have been presumed to be due to lipid phase transitions. In the adrenal microsomes, a van 't Hoff plot of the apparent substrate dissociation constant exhibiting two discontinuities, which correlated with lipids' phase transition temperatures * was reported previously [4] from this laboratory. In the present study, the effect of temperature on the overall steroid C-21 hydroxylation of 17a-hydroxyprogesterone has been investigated in the adrenal microsomes. In addition, the temperature dependency of the substrate-cytochrome binding reaction is investigated under turnover conditions (NADPH + 02). The results indicate that thermally induced transitions observed for the substrate dissociation constant as well as those for the fluidity of the membrane lipids are distinct from but not necessarily independent of the single transition observed for the overall enzyme activity. A possible thermodynamic relationship between the substrate-cytochrome P-450 binding reaction and substrafe hydroxylation is pointed out. Materials and Methods

Chemicals. Bovine serum albumin, glucose 6-phosphate; glucose-6-phosphate dehydrogenase, NADP and 17a-hydroxyprogesterone, were purchased from Sigma Chemical Co. Triton X-114 was from Rohm and Haas Co., and 1,6diphenyl-l,3,5-hexatriene and tetrahydrofuran from Aldrich Chemical Co., RNAase-free sucrose from Schwartz/Mann Co. Preparation of the microsomal fraction. The procedure for homogenization of the cortex tissue and isolation of the microsomal fraction was exactly as * T h e t e m p e r a t u r e s w h i c h m a r k the b e g i n n i n g and the e n d o f m e l t i n g o f lipids in lateral phase s e p a r a t i o n p r o c e s s have b e e n referred to as lipid p h a s e t r a n s i t i o n t e m p e r a t u r e s [ 4 , 1 0 ] ~ lipid phase transition b o u n daries [ 2 0 ] and characteristic t e m p e r a t u r e s of t h e lipids' p h a s e t r a n s i t i o n [ 2 1 , 2 2 ] . In this paper t h e y will be referred to as c h a r a c t e r i s t i c t e m p e r a t u r e s o f the lipid p h a s e t r a n s i t i o n .

383 described before [4] except that a slightly higher concentration (0.3 M) and RNAase-free sucrose containing 1 mM EDTA and 0.0025 M Hepes buffer, pH 7.4, was used as the medium for homogenizing the tissue. The microsomal fraction was further fractionated by sucrose density gradient (1.2 M; 0.9 M and 0.4 M) centrifugation also as described previously [4]. The band which was collected between 0.9 M and 1.2 M sucrose was used in the experiments. All preparations were stored at --68°C until use. Determination of the apparent substrate dissociation constant, K~. The substrate-produced Type I difference spectrum of cytochrome P-450 characterized by a trough at 421 nm, at peak at 388--390 nm and an isosbestic point at 407 nm was used as the criterion for the binding of the substrate to the cytochrome. The assay system and the procedure for equilibrating the assay system at different temperatures, titrating with the substrate, recording of the absorbancy change (L~4407_421nm)and calculation of the dissociation constant have been described previously in detail [4]. Under the present technical and experimental conditions, the standard error of the mean of five Ks values calculated from five identical titrations expressed as percent of the mean is 1.3. The standard error of the technique of adding the substrate determined by ten identical additions of cytochrome c to a buffer and recording the absorption is 0.8% of the mean. Measurement of the Type I spectral change under turnover conditions (NADPH + 02 ). The reaction mixture consisted of 2.6 ml of glycylglycine buffer, pH 7.4, containing 1.6% dialyzed bovine serum albumin, 120 mM NaC1, 24 mM glycylglycine, 4.8 mM KC1 and 0.9 mM MgC12, 0.2 ml of NADPH generating system (0.6 mM NADP + 3.3 mM glucose 6-phosphate + 0.2 units of glucose-6-phosphate dehydrogenase) and 0.2 ml (0.8--1.0 mg protein) of the microsomal preparation. This reaction mixture was contained in a photometric cuvette. After temperature equilibration and equilibration with the NADPH generating system, a baseline B (Fig. 1A) of equal output of the two photomultipliers (k, find k2) was recorded. The reaction was started by adding, as previously described [4], 0.4 or 0.6 pl of a methanolic solution of 17a-hydroxyprogesterone at the indicated point (Fig. 1A). The decrease in absorbance which is indicative of formation of the Type I spectral change as well as its disappearance was recorded as a function of time. In this type of experiment, in addition to the precision with which the substrate had to be added, it was important to consider the following points: (1) the time required for mixing of the added substrate; (2) the fraction of the substrate-produced spectral change which would have disappeared during the mixing time; (3) the absence of possible nonspecific absorption changes contributing to the disappearance of the spectral change must be ascertained. The mixing time of the substrate was determined in the absence uf NADPH generating system by measuring the time required to attain maximum AA407-421n m after the addition of the substrate. Under the present experimental conditions, approx. 90% for the spectral change could be observed within 5 s. The time required for maximum change was between 10 and 15 s. The substrate-produced spectral change under turnover conditions was corrected graphically for the fraction of the AA407_421n m which disappeared during the mixing time. This was accomplished by extrapolating the initial rate

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T (°C) Fig. 1. S u b s t r a t e - p r o d u c e d T y p e I s p e c t r a l c h a n g e u n d e r t u r n o v e r c o n d i t i o n s : A. R e c o r d i n g of A A 4 0 7 _ 4 2 1 n m w i t h t h e dual w a v e l e n g t h filter p h o t o m e t e r . T h e r e a c t i o n m i x t u r e ( g l y c y l g l y c i n e b u f f e r plus N A D P H g e n e r a t i n g s y s t e m ) c o n t a i n e d 0.3 m g of m i c r o s o m a l p r o t e i n p e r ml. T i m e scale f r o m r i g h t to left. T h e details of p r o c e d u r e a n d the r e a c t i o n m i x t u r e are d e s c r i b e d a b o v e ( M e t h o d s , p. 3 8 3 ) . B. A A 4 0 7 _ 4 2 1 n m as a f u n c t i o n of t e m p e r a t u r e : × × × , AA o b s e r v e d u n d e r t u r n o v e r c o n d i t i o n s ; l e e , AA graphically c o r r e c t e d as d e s c r i b e d a b o v e ; oo©, AA o b s e r v e d in t h e a b s e n c e of N A D P H g e n e r a t i n g s y s t e m . C. R e p e a t e d s c a n n i n g o f t h e s u b s t r a t e - p r o d u c e d d i f f e r e n t s p e c t r u m w i t h a DW-2 r a p i d s c a n n i n g s p e c t r o p h o t o m e t e r . T h e e x p e r i m e n t a l a n d t h e r e f e r e n c e c u v e t t e s c o n t a i n e d 3.0 m l each of the r e a c t i o n m i x t u r e (1.5 m g m i c r o s o m a l p r o t e i n p e r m l ) . A p p r o x i m a t e l y 10 pM 17c~-OH p r o g e s t e r o n e was a d d e d to t h e e x p e r i m e n t a i c u v e t t e a n d t h e d i f f e r e n c e s p e c t r u m was r e c o r d e d r e p e a t e d l y b e t w e e n 3 6 0 a n d 4 8 0 n m .

of formation of the spectral change and the rate of disappearance of the spectral change after 15 s after the addition of the substrate to intersect at a point (Fig. 1A). The absorbance difference between the baseline and the point of intersection was taken as true AA. The validity of the graphically corrected AA was tested experimentally by measuring the spectral change in the absence of reducing agent. The results are shown in Fig. lB. The graphically corrected AA (dots) lie only slightly higher than those determined experimentally (circles). The Ks values calculated from the graphically corrected AA are 0.46--0.5% underestimated. Since this figure is less than the standard error of determination of K s by the titration technique and of addition of the substrate (Methods), it is considered insignificant.

385 Determination of the fluidity of the microsomal membranes. The fluidity was determined as described previously [4] by the technique of fluorescence polarization :of the hydrophobic probe 1,6-diphenyl-l,3,5-hexatriene according to Shinitzky and Inbar [13]. Their method was followed for fluorescent labelling of the microsomal membranes ( 0 . 4 - 0 . 5 mg protein per ml; 0.5 pM diphenylhexatriene) in saline-containing 0.005 M sodium phosphate buffer, pH 7.4. The measurements were made using an Elscint microviscosimeter which is equipped to measure polarization directly as a function of temperature. According to specifications, the accuracy of polarization values expected with this instrument is -+1.15%. The reproducibility of the polarization values appears to be 100%. The temperature was measured as before with a copper constant thermocouple. Microviscosity was calculated from polarization values according to Shinitzky and Inbar [13]. Results

The effect of temperature on the C-21 hydroxylation of 17a-OH progesterone The results of a representative experiment are shown in Fig. 2 (section A). The figure shows the Arrhenius plot of the rate of hydroxylation in the presence of excess of the substrate (200 pM). The rate was determined at intervals of 2--3 deg.C). Under these conditions, the Arrhenius plot exhibits a single break at 23.6°C (24.2 + 1.6°C, n = 3). The apparent activation energies are 18.6 kcal/mol (20.9 _+3.9 kcal/mol, n = 3) and 32.3 kcal/mol (34.6 _+4.8 kcal/mol, n = 3) on the high and the low temperature sides of the break, respectively.

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386 Since similar breaks in Arrhenius activity plots of membrane enzymes have been attributed to lipid phase transitions, the effect of a lipid perturbing agent on the temperature dependency of the hydroxylation was studied. The addition of the non-ionic detergent Triton X-114 was without any significant effect on either the position of the break or the activation energies (Fig. 2, section B). In order to find out whether or not the single break observed in the Arrhenius plot of the enzyme activity is distinct from the breaks observed in the van't Hoff plot of the substrate dissociation constant, the concentration of the detergent tested was that which altered strikingly the temperature profile of the substrate dissociation constant. In the presence of the detergent, the van't Hoff plot of K s exhibit only the break at 21°C [4]. Doubling the concentration of the detergent was also without effect on the temperature profile of the hydroxylation. In the absence as well as in the presence of the detergent, the activation energies are considerably higher than those observed at low substrate concentration (Fig. 3). It is possible that high concentration of the lipid soluble substrate has altered noncovalent interactions such as lipid-lipid and lipid-protein interactions.

The effect of temperature on the disappearance of 17a-hydroxyprogesteroneproduced type I spectral change under turnover conditions The Type I spectral change produced by hydroxylatable steroids disappear under turnover conditions (p. 384) as the substrate is converted to its hydro-

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387 xylated product. That this disappearance is associated with product formation has been demonstrated in adrenocortical mitochondria, in the case of 11-/3 hydroxylation of deoxycorticosterone [14] and the oxidation of 20a-hydroxycholesterol [15] and in adrenal microsomes in the case of C-21 hydroxylation of 17a-hydroxyprogesterone [16,17]. Therefore, the disappearance of the Type I spectral change provides a convenient method to study the reaction at low substrate concentration. The results of a representative experiment using 0.1134 pM 17a-hydroxyprogesterone are shown in Fig. 3 in the form of an Arrhenius plot of reciprocal of time required for half of the spectral change to disappear (Tl/2). The absorbance change after the addition of the substrate was corrected as described under Methods. The uncorrected values were 10--15% lower. However, the temperature profile of the disappearance half-time was essentially the same before and after correction. Similar to the case when the hydroxylated product was determined (Fig. 2), the Arrhenius plot of T1/2 (Fig. 3A) exhibited a single break at 24.9°C (24.1 + 0.5°C, n = 4). The activation energies on the low and hightemperature side of the break are 13.5 + 1.55 kcal/mol and 6.18 + 2.16 kcal/mol (n = 4), respectively. Although the temperature at which the break occurs is nearly the same as that when product formation was determined in the presence of high substrate concentration, the activation energies are considerably lower. It is pointed out that Arrhenius plots in the case of enzymes with temperature-dependent Km can exhibit erroneous breaks if activity is essayed at fixed low substrate concentration [18]. In the present experiments, in addition to the position of the break, the shape of the Arrhenius plot of the 1/T1/2 is essentially the same as that observed in the presence of high concentration of the substrate. Furthermore, when TI/2 was determined at two different concentrations of the substrate at each of four different temperatures between 10 and 40°C, it was found to be independent of substrate concentration. In other words, it is independent of the concentration of the substrate-bound cytochrome P-450. Therefore, decrease in the concentration of the substrate-bound cytochrome P-450 with increase in temperature due to increase in K s should not interfere with the temperature profile of Tz/2. The temperature profile of T~/2 is also similar to that in the case where product formation was determined at high substrate concentration, in that the detergent Triton X-114 was essentially without effect on the position of the break as welt as on the activation energies (Fig. 3B).

The effect of temperature on the apparent dissociation constant under turnover conditions In previously reported studies [4] on the effect of temperature on the substrate dissociation constant, two breaks (18 and 31°C) in the van't Hoff plot of K S were detectable with certainty. In those experiments, the dissociation constant was determined by a semi-micro titration technique [4] at intervals of 1.5 to 2.5 deg.C. Subsequent more detailed study of the temperature profile by improved titration technique and careful choice of the temperature intervals revealed at least four breaks (18.3 + 1.9°C; 24.7 +_2.2°C; 31.6 + 1.7°C and 37 + 0.8°C, n = 3) between 10 and 40°C (Narasimhulu, in preparation). In the present experiments, the temperature dependency of the substrate dissociation constant is investigated under turnover conditions as follows. The magnitude

388 of AA407-421nm, which is a function of the substrate dissociation constant at a given substrate concentration was determined as a function of temperature. The dissociation constant was calculated according to the following equation:

where S = concentration of 17c~-hydroxyprogesterone; 2~4max = observed Z~407_421nm at saturating concentration of the substrate determined at all temperatures by adding at least 20 × Ks. There was no significant difference in AAmaxS at different temperatures; AA = observed AA,~ov___421n m corrected as described under Methods. The results of a representative experiment are shown in Fig. 4. K s is plotted as a function of reciprocal temperature on the centrigrade scale (section A). K~S determined by titration in the absence of reducing agent (Section A, x x ) are nearly equal to the calculated KsS (section A, dots). Section B

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I ToC Fig. 4. T e m p e r a t u r e d e p e n d e n c y o f t h e a p p a r e n t s u b s t r a t e d i s s o c i a t i o n c o n s t a n t u n d e r t u r n o v e r c o n d i t i o n s . T h e d i s s o c i a t i o n c o n s t a n t w a s c a l c u l a t e d f r o m t h e r e s u l t s o f t h e s a m e e x p e r i m e n t as t h a t r e p o r t e d in F i g . 3. A A 4 0 7 - - 4 2 1 n m w a s m e a s u r e d as d e s c r i b e d u n d e r M e t h o d s . T h e p r o c e d u r e f o r c a l c u l a t i o n o f K s is d e s c r i b e d in t h e t e x t ( e q u a t i o n ) . S e c t i o n A d o t s r e p r e s e n t c a l c u l a t e d Ks; C r o s s e s r e p r e s e n t K s d e t e r m i n e d f r o m t h e r e s u l t s of d i r e c t t i t r a t i o n in t h e a b s e n c e o f N A D P H . S e c t i o n B. V a n ' t H o f f Plot. T h e A H v a l u e s w e r e o b t a i n e d f r o m t h e l e a s t s q u a r e c a l c u l a t i o n of t h e slope a n d t h e i n t e r c e p t . C o r r e l a t i o n c o e f f i c i e n t s f o r all t h e s e g m e n t s are 0 . 9 9 1 3 to 0 . 9 9 8 2 e x c e p t f o r t h e s e g m e n t b e t w e e n 22 a n d 1 7 ° C , w h i c h is 0 . 9 6 2 1 . T h e m a x i m u m v a r i a t i o n o f t h e A H v a l u e s d u e to 1 . 3 % s t a n d a r d e r r o r o f d e t e r m i n a t i o n of K s is 1 2 - - 1 6 % in all t e m p e r a t u r e r e g i o n s .

389 shows the van't Hoff plot. The four breaks (37; 31, 25 and 17.3°C) are also detectable under turnover conditions. The enthalpies for different segments (indicated in Fig. 4B) are strikingly different for different temperature regions. The free energy (AG) ranged between --9.4 and --9.54 kcal/mol between 10 and 40°C. The AG value is reproducible within +0.6 kcal/mol in different batches of microsomes. But the enthalpies, although reproducible within the same batch, varied considerably from batch to batch. The reason for this variation is not known. It is reported [19] that changes in lipid composition such as cholesterol-to-phospholipid ratio in biological membranes alter the microviscosity of the membranes. Information from the slaughter house indicates that different batches of adrenal glands are composed of glands from different proportions of steers from different ages. However, the lipid composition of different batches of the microsomes remains to be determined. In experiments such as that reported in Fig. 4, the substrate-produced spectral change as well as the disappearance of the spectral change could be measured under turnover conditions in one and the same experiment. This provided an opportunity to compare the apparent thermodynamic parameters for the substrate dissociation constant with those of the overall enzyme activity under identical conditions. The thermodynamic parameters at physiologic temperature for the substrate dissociation constant are as follows: AH =--18.7 kcal/mol; AG = --9.46 kcal/mol and AS = 29.9 cal/K per mol. At physiologic temperature, the apparent activation energy for the overall enzyme activity is 6.6 kcal/mol. This is the activation energy of hydroxylation at low substrate concentration. The effect o f temperature on the fluidity of the microsomal membranes The technique is described under Methods. In previously reported experi0.40

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390 ments [4] fluorescence polarization was calculated from fluorescence intensities measured at intervals of every 2 - 3 deg.C, using a Perkin-Elmer fluorescence spectrometer with a Hitachi polarizer accessory. Under these conditions, two breaks (22 and 32°C) in the temperature profile of the fluidity between 11 and 40°C were detectable. In the present experiments, fluorescence polarization measurements are made at intervals of 1 deg, C or less using an Elscint fluorescence instrument which allows measurement of polarization directly with high degree of reproducibility and accuracy (Methods). Under these conditions, four breaks were always detectable between 11 and 40°C. In some microsomal preparations an additional break close to 10°C was observed. The results of a representative experiment are shown in Fig. 5. The figure shows the logarithm of microviscosity as a function of reciprocal absolute temperature. Four breaks (17, 24.5, 30.6 and 37.2°C) detected are nearly at the same temperature as those found in the van't H o f f plot of the substrate association constant. The microsomal preparation used in this experiment is the same batch as that used for the experiment reported in Fig. 4. These temperatures varied (18.04 + 1.4; 24.8 + 1.6; 31.4 + 0.6 and 36.4 + 0.5°C, n = 5) from batch to batch of the microsome preparation. The variation was in the same direction as that observed in the case of Ks. The enthalpies for lipid fusion also varied from preparation to preparation. The range of values for different segments 40--37; 37--31; 31--25 and 25--17°C are 6 . 0 2 + 1.95; 5.36 + 1.18; 503 + 0.62 and 4.51 + 0.52 kcal/mol (n = 5). Discussion Functional correlations of lipid phase transitions in biological membranes have been demonstrated in bacterial systems [3,22] and recently in a mammalian system [20] in endoplasmic reticulum membranes of cultured animal cells. In the mammalian system, ESR analysis of the membranes has revealed four characteristic temperatures (16, 22, 32 and 38°C) of lipid phase transition. In addition, two membrane processes (transport processes) which should be catalyzed by components that span the thickness of the membrane are affected at all of the characteristic temperatures. From physical and physiologic evidence, Wisnieski et al. [2] have suggested that the four characteristic temperatures are attributable to two independent lateral phase separation of lipids in the inner and the outer half of the bilayer with an asymmetric lipid distribution. In the present study, the four characteristic temperatures (17, 25, 31 and 37°C) of the lipid phase transition as detected by the technique of fluorescence polarization of the hydrophobic probe 1,6-diphenyl-l,3,5-hexatriene are very close to those reported for the endoplasmic reticulum membranes of cultured animal cells [ 20]. The apparent substrate dissociation constant is affected at all of the characteristic temperatures, as indicated by the temperature breaks in the van't Hoff plot of Ks. If the four breaks are sufficient to account for the two monolayers [20], the components involved in the substrate-cytochrome P-450 binding reaction must be under the control of the entire thickness of the membrane. The overall enzyme activity must then be under the control of a limited region of the bilayer since the Arrhenius activity plot exhibits a single break. The lack of effect of the detergent Triton X-114 on the single break

~391

while the detergent eliminated the low temperature breaks in the case of K s as well as the fluidity [4] suggests that the factors responsible for the single break in the case of enzyme activity are distinct from, but not necessarily independent of, those responsible for the multiple breaks observed in the case of Ks. The lack of effect of the detergent also suggests the possibility that the events responsible for the single break are not mediated through lipids to any large extent. It is also possible that under the present experimental conditions, the lipid which may influence the enzyme activity may not be accessible to the detergent. In biological membranes, interpretation of the effects of temperature and other perturbations is very complex. However, the effects of temperature on the substrate-cytochrome P-450 binding reaction under turnover conditions and its relationship to the overall hydroxylation can be visualized in terms of the present concepts of membrane dynamics at least as follows. As has been suggested for tightly bound membrane enzymes such as cytochrome bs [23] and cytochrome oxidase [24], cytochrome P-450 is probably lodged in the membrane by hydrophobic interactions with alkyl chains of the phospholipids. The membrane could then serve to trap the hydrophobic substrate and allow it to diffuse within the lipid phase to the binding site on the enzyme [25]. However, the affinity of the steroid cannot be governed by any difusion limited process because the affinity increases with decrease in temperature and the diffusion of steroids is drastically decreased below the lipids' phase transition temperatures [25]. A second possible explanation for the temperature dependancy of K s is that the cytochrome could assume different conformations dependding upon the fluidity of the lipid environment with different affinities to the substrate. If the lipid-protein interactions are strong, as would be expected for intrinsic membrane proteins such as cytochrome P-450, conformation of the protein may be drastically altered by changes in the fluidity [26]. As indicated earlier, it is suggested that thermally induced conformation transitions required for the functioning of membrane enzymes are mediated through the lipids. In the present experiments, the free energy o f - - 9 . 4 to - 9 . 5 6 kcal/mol found for the substrate-cytochrome P-450 binding reaction is sufficiently large to be compatible with conformation change. The magnitude of the free energy is comparable to the values reported for non-covalent protein-protein [27] or protein-hydrocarbon [28] interactions. At physiologic temperature, in addition to the substantial negative freee energy (--9.5 kcal/mol), the substrate-cytochrome binding occurs with negative enthalpy (AH = --18 kcal/mol) and decrease in entropy (AS = --29.9 cal/K per mol). At physiologic temperature and low concentration of substrate, the activation energy for the overall hydroxylation reaction is 6.6 kcal/mol. Thus the magnitude of the free energy of the substrate-binding reaction is more than sufficient to account for the apparent activation energy of the overall enzyme reaction. Therefore, the substrate-cytochrome binding reaction has the potential of being a source of energy for the endergonic steps in the hydroxylation reaction. However, the mechanism whereby the energy may be utilized in the reaction can only be speculative at the present time. In connection with the electron transport associated with mitochondrial energy metabolism, it is hypothesized that energy is captured, transmitted and used by protein conformation

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changes [29]. In addition, possibility of oxidation-reduction by conformationally induced change in the oxidation-reduction potential at the catalytic site has been suggested [29]. Such a mechanism may also be applicable for oxidation-reduction reactions associated with steroid hydroxylation. Substratedependent low-to-high potential transition has been reported for bacterial cytochrome P-450 [30] and remains to be demonstrated in the adrenal system. The possibility of the substrate-cytochrome binding reaction being a source of energy for the overall enzyme activity is consistent with the results reported previously [16,17]. The results suggested that the substrate-produced Type I spectral change is associated with activation of the P-450 reduction mechanism independent of substrate hydroxylation and oxygen activation. This implied that the spectral change may represent some type of regulatory mechanism for electron transport. The Type I substrate-dependent enhancement in the reduction of cytochrome P-450 under conditions which inhibited hydroxylation has also been reported for hepatic microsomes [31]. That such a substrateinitiated event could be the site of primary control of steroidogenesis has been suggested in the case of 11-/3 hydroxylation in adrenal cortex mitochondria. Acknowledgements This work is supported by O.N.R. Contract N00014-75-C-0322 and N.I.H. grant AM 18545. The author thanks Ms. Amy Yen for her excellent technical assistance. References 1 Linden, C.D., Wright, K.L., McConnell, H.M. and Fox, C.F. (1973) Proc. Natl. Acad. Sci. 70, 2271-2275 2 Wisnleski, B.J., Parkes, J.G., Huang, Y.O. and Fox, C.F. (1974) Proe. Nat]. Acad. Sci. 71, 4381-4385 3 MeElhaney, R.N. (1974) J. S u p r e m o l . Struct. 2 , 6 1 7 - - 6 2 8 4 Narasimhulu, S. (1977) B i o c h i m . B i o p h y s . Acta 4 8 7 , 3 7 8 - - 3 8 7 5 Raison, J.K. (1973) Bioenergeties 4, 285--309 6 Levin, W., Ryan, D., West, S. and Lu, A.Y.H. (1974) J. Biol. Chem. 249, 1747--1754 7 Narasimhulu, S. (1974) Drug Metab. and D i s p o s i t i o n 2, 573--576 8 Peterson, J.A., Ebel, R.E., O'Keefe, D.H., Matsubara, T. and E s t a b r o o k , R.W. (1976) J. Biol. Chem. 251, 4010--4016 9 Duppel, W. and Ullrich, V. (1976) Biochim. Biophys. Aeta 4 2 6 , 3 9 9 - - 4 0 7 10 Stier, A. and Sackman, E. (1973) Biochim. B i o p h y s . A e t a 3 1 1 , 4 0 0 - - 4 0 8 11 Rosenthal, O. and Narasimhulu, S. (1969) M e t h o d s in E n z y m o l o g y (Clayton, R.B., ed.), Vol. 15, Pp. 596--638, A c a d e m i c Press, N e w York 12 Peterson, R.E., Karrer, A. and Guerra, S.L. (1957) A n a l Chem. 29, 144--149 13 Shinitzky, M. and Inbar, M. (1974) J. Mol. Biol. 85, 6 0 3 - 6 1 5 14 Oldham, S.B., Wilson, L.D., Landgrof, W.K. and Harding, B.W. (1968) Arch. B i o c h e m . B i o p h y s . 123, 484--495 15 Wilson, L.D. and Harding, B.W. (1973) J. Biol. Chem. 248, 9--14 16 Naraaimhulu, S. (1971) Arch. Biochem. Biophys. 147, 391--404 17 Narasimhulu, S, (1971) Arch. Biochem. Biophys. 1 4 7 , 3 8 4 - - 3 9 0 18 Silvius, J.R., Read, B.D. and M c E l h a n e y , R.N. (1978) 1 9 9 , 9 0 2 - - 9 0 4 19 Shinitzky, M. and Inbax, M. (1976) Bioehim. B i o p h y s . A c t a 433, 133--149 20 Wisnieski, B.J., Huang, Y.O. and Fox, F.C. (1974) J. S u p r a m o l . Struct. 2, 593--608 21 Kleerman, W., Grant, C.W.M. and McConnell, H.M. (1974) J. S u p r a m o l . Struct. 2, 6 0 9 - 6 1 6 22 Lindin, C.D. and Fox, G.F. (1973) J. Supramol. Struet. 1, 535--544 23 Spatz, L. and Strittmatter, P. (1971) Proc. Natl. A e a d . Sei. U.S. 68, 1042--1046 24 Vanderkooi, G. (1974) Bioehim. B i o p h y s . A c t a 3 4 4 , 3 0 7 - - 3 4 5

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Devaux, P. and McConnell, H.M. (1972) J. Am. Chem. Soc. 94, 4 4 7 5 - - 4 4 8 1 Sullivan, K., Jain, M.K. and Koch, A.L. (1974) Biochim. Biophys. Acta 352, 287--297 Klotz, I.M., Langen~aan, N.R. and Darnall, D.W. (1970) Annu. Rev. Biochem. 39, 25--62 Tanford, C. (1973) The Hydrophobic Effect, p. 132, J. Wiley and Sons, New Y ork Boyer, P.D. (1974) in Dynamics of Energy-Transducing Membranes (Ernster, L., Estabrook, R.W. and Slater, E.C., eds.), The B.B.A. Library, Vol. 13, pp. 289--301, Elsevier, A ms t e rda m Gunzalus, I.C., Marshall, V.P., Meeks, J.R. and Lipscomb, J.D. (1973) in 9t h Int. Congr. Bioehem. S t o c k h o l m Syrup., 7Sc Section, 7 Gigon, P.L., Gram, T.E. and Gillette, J.R. (1969) Mol. Pharmacol. 5, 109--122 Harding~ B.W., Bell, J.J., Wilson, L.D. and Whysner, J.A. (1969) Adv. Enzyme Regul. 7 , 2 3 7 - - 2 5 7 Narasimhulu, S. (1977) Fed. Proc. 37, Abst. 2071